Pichia pastoris strains for producing predominantly homogeneous glycan structure

Abstract

Disclosed herein are novel Pichia pastoris strains for expression of exogenous proteins with substantially homogeneous N-glycans. The strains are genetically engineered to include a mutant OCH1 allele which is transcribed into an mRNA coding for a mutant OCH1 gene product (i.e., α-1,6-mannosyltransferase, or “OCH1 protein”). The mutant OCH1protein contains a catalytic domain substantially identical to that of the wild type OCH1 protein, but lacks an N-terminal sequence necessary to target the OCH1 protein to the Golgi apparatus. The strains disclosed herein are robust, stable, and transformable, and the mutant OCH1 allele and the ability to produce substantially homogeneous N-glycans are maintained for generations after rounds of freezing and thawing and after subsequent transformations.

What is claimed is: 
     
         1 . A purified preparation of a protein made by a method which comprises:
 a. expressing the protein from an engineered stable strain of  Pichia pastoris  in a cell culture, wherein the strain comprises a mutant OCH1 allele comprising a nucleotide sequence encoding a mutant OCH1 protein, wherein said mutant OCH1 protein (i) comprises a catalytic domain comprising amino acid residues 45-404 of the wild type OCH1 protein of the amino acid sequence of SEQ ID NO: 2, or (ii) comprises a catalytic domain comprising at least 90% amino acid sequence identity to the amino acid residues 45-404 of the wild type OCH1 protein of the amino acid sequence of SEQ ID NO: 2, and wherein said mutant OCH1 protein has α-1, 6-mannosyltransferase activity, and wherein said mutant OCH1 protein lacks an N-terminal sequence for targeting the mutant OCH1 protein to the Golgi apparatus; and   b. isolate the protein from the cell culture to obtain the purified preparation of the protein, wherein the purified preparation is substantially homogeneous in the N-glycan structures, with Man5GlcNac2 constituting at least 80% of the N-glycan structures.   
     
     
         2 . The purified preparation of a protein of  claim 1 , wherein the protein is selected from the group consisting of trastuzumab,  Candida antarctica  lipase A,  Candida antarctica  lipase B, and human serum transferrin. 
     
     
         3 . The purified preparation of a protein of  claim 1 , wherein Man5GlcNac2 constitutes at least 85% of the N-glycan structures in the purified preparation of the protein. 
     
     
         4 . The purified preparation of a protein of  claim 1 , wherein Man5GlcNac2 constitutes at least 90% of the N-glycan structures in the purified preparation of the protein. 
     
     
         5 . The purified preparation of a protein of  claim 1 , wherein the catalytic domain comprises at least 95% amino acid sequence identity to the amino acid residues 45-404 of the amino acid sequence of SEQ ID NO: 2. 
     
     
         6 . The purified preparation of a protein of  claim 1 , wherein the mutant OCH1 protein lacks a membrane anchor domain at the N-terminal region. 
     
     
         7 . The purified preparation of a protein of  claim 1 , wherein said mutant OCH1 protein comprises the amino acid sequence of SEQ ID NO: 3. 
     
     
         8 . The purified preparation of a protein of  claim 1 , wherein said engineered stable strain further comprises a nucleic acid encoding for and expressing an α-1,2-mannosidase. 
     
     
         9 . The purified preparation of a protein of  claim 8 , wherein said nucleic acid coding for and expressing said α-1,2-mannosidase is integrated at the OCH1 locus of the engineered stable strain. 
     
     
         10 . The purified preparation of a protein of  claim 9 , wherein the OCH1 locus comprises the nucleotide sequence of SEQ ID NO: 1. 
     
     
         11 . A purified preparation of a protein, wherein the protein is selected from the group consisting of trastuzumab,  Candida antarctica  lipase A,  Candida antarctica  lipase B, and human serum transferrin, and wherein the purified preparation is substantially homogeneous in the N-glycan structures, with Man5GlcNac2 constituting at least 80% of the N-glycan structures. 
     
     
         12 . The purified preparation of a protein of  claim 11 , wherein Man5GlcNac2 constitutes at least 85% of the N-glycan structures in the purified preparation of the protein. 
     
     
         13 . The purified preparation of a protein of  claim 11 , wherein Man5GlcNac2 constitutes at least 90% of the N-glycan structures in the purified preparation of the protein. 
     
     
         14 . A method for obtaining a purified preparation of a protein, comprising:
 a. expressing the protein from an engineered stable strain of  Pichia pastoris  in a cell culture, wherein the strain comprises a mutant OCH1 allele comprising a nucleotide sequence encoding a mutant OCH1 protein, wherein said mutant OCH1 protein (i) comprises a catalytic domain comprising amino acid residues 45-404 of the wild type OCH1 protein of the amino acid sequence of SEQ ID NO: 2, or (ii) comprises a catalytic domain comprising at least 90% amino acid sequence identity to the amino acid residues 45-404 of the wild type OCH1 protein of the amino acid sequence of SEQ ID NO: 2, and wherein said mutant OCH1 protein has α-1, 6-mannosyltransferase activity, and wherein said mutant OCH1 protein lacks an N-terminal sequence for targeting the mutant OCH1 protein to the Golgi apparatus; and   b. isolating the protein from the cell culture to obtain the purified preparation of the protein, wherein the purified preparation is substantially homogeneous in the N-glycan structures, with Man5GlcNac2 constituting at least 80% of the N-glycan structures.   
     
     
         15 . The method of  claim 14 , wherein the protein is selected from the group consisting of trastuzumab,  Candida antarctica  lipase A,  Candida antarctica  lipase B, and human serum transferrin. 
     
     
         16 . The method of  claim 14 , wherein Man5GlcNac2 constitutes at least 85% of the N-glycan structures in the purified preparation of the protein. 
     
     
         17 . The method of  claim 14 , wherein Man5GlcNac2 constitutes at least 90% of the N-glycan structures in the purified preparation of the protein. 
     
     
         18 . The method of  claim 14 , wherein the mutant OCH1 protein lacks a membrane anchor domain at the N-terminal region. 
     
     
         19 . The method of  claim 14 , wherein said mutant OCH1 protein comprises the amino acid sequence of SEQ ID NO: 3. 
     
     
         20 . The method of  claim 14 , wherein said engineered stable strain further comprises a nucleic acid encoding for and expressing an α-1,2-mannosidase.